Aims: To establish the biotechnology platforms for production of bio-based chemicals in various micro-organisms is considered as a promising target to improve renewable production of isoprene. Methods and Results: In this study, we heterologously expressed the mevalonate (MVA) isoprene biosynthesis pathway, and explored three strategies of increasing isoprene production in Escherichia coli. We first manipulated the expression levels of the MVA pathway genes through changing the gene cassettes and promoters. To introduce cofactor engineering, we then overexpressed NADP-dependent glyceraldehyde-3-phosphate dehydrogenase gene from Clostridium acetobutylicum to supply available NADPH. To reduce the inhibitory by-product accumulation, we finally knocked out acetate-producing genes, phosphate acetyl transferase and pyruvate oxidase B in E. coli JM109 (DE3), decreasing acetate accumulation 89% and increasing isoprene production 39%. The strategies described here finally increased the isoprene titre to 92 mg l À1 in two-gene deletion strain JMAB-4T7P1Trc, increasing 2Á6-fold comparing to strain JM7T7. Conclusion: The multimodularly engineering approaches including promoter engineering, cofactor engineering and by-product reducing could be used to improve isoprene production in E. coli. Significance and Impact of the Study: The metabolic strategies in this study show us directions for further studies to promote transformation of renewable sources to isoprene.

Aims: Poly-c-glutamic acid (c-PGA) is an excellent water-soluble biosynthesis material. To confirm the rate-limiting steps of c-PGA biosynthesis pathway, we introduced a heterologous Bacillus strain pathway and employed an enzymemodulated dismemberment strategy in Escherichia coli. Methods and Results: In this study, we heterologously introduced the c-PGA biosynthesis pathway of two laboratory-preserved strains-Bacillus amyloliquefaciens FZB42 and Bacillus subtilis 168 into E. coli, and compared their c-PGA production levels. Next, by changing the plasmid copy numbers and supplying sodium glutamate, we explored the effects of gene expression levels and concentrations of the substrate L-glutamic acid on c-PGA production. We finally employed a two-plasmid induction system using an enzyme-modulated dismemberment of pgsBCAE operon to confirm the ratelimiting genes of the c-PGA biosynthesis pathway. Conclusion: Through heterologously over-expressing the genes of the c-PGA biosynthesis pathway and exploring gene expression levels, we produced 0Á77 g l À1 c-PGA in strain RSF-EBCAE(BS). We also confirmed that the ratelimiting genes of the c-PGA biosynthesis pathway were pgsB and pgsC. Significance and Impact of the Study: This work is beneficial to increase c-PGA production and study the mechanism of c-PGA biosynthesis enzymes.

Co-localizing biochemical processes is a great strategy when expressing the heterologous metabolic pathway for product biosynthesis. The RNA scaffold is a flexible and efficient synthetic compartmentalization method to co-localize the enzymes involved in the metabolic pathway by binding to the specific RNA, binding domains fused with the engineered enzymes. Herein, we designed two artificial RNA scaffold structures�0D RNA scaffolds and 2D RNA scaffolds�using the reported aptamers PP7 and BIV-Tat and the corresponding RNA-binding domains (RBDs). We verified the interaction of the RBD and RNA aptamer in vitro and in vivo. Then, we determined the efficiencies of these RNA scaffolds by co-localizing fluorescent proteins. We employed the RNA scaffolds combined with the enzyme fusion strategies to increase the metabolic flux involved in the enzymes of the mevalonate pathway for mevalonate and isoprene production. Compared with the no RNA scaffold strain, the mevalonate levels of the 0D RNA scaffolds and 2D RNA scaffolds increased by 84.1% (3.13 ± 0.03 g/L) and 76.5% (3.00 ± 0.09 g/L), respectively. We applied the 0D RNA scaffolds for increasing the isoprene production by localizing the enzymes involved in a heterologous multi-enzyme pathway. When applying the RNA scaffolds for co-localizing the enzymes mvaE and mvaS, the isoprene production reached to 609.3 ± 57.9 mg/L, increasing by 142% compared with the no RNA scaffold strain. Our results indicate that the RNA scaffold is a powerful tool for improving the efficiencies of the reaction process in the metabolic pathway.